Hyaluronic acid is a natural, linear polysaccharide, biocompatible and biodegradable, constituted by a repeating disaccharide unit formed by glucuronic and N-acetylate glucosamine linked by glycoside bonds .beta. 1-3 and .alpha. 1-4.
Hyaluronic acid is present in the connective tissues of higher organisms, in the synovial fluid, in the umbilical cord and in cockscombs; it can also be synthesized from certain bacterial forms such as Streptococci (Kendall et al, Journ. Biol. Chem., vol. 118, page 61, 1937).
Hyaluronic acid plays a vital role in many biological processes, such as tissue hydration, proteoglycan organization, cell differentiation and angiogenesis. There are many biomedical applications linked with the Theological properties of hyaluronic acid solutions: one important sector is that of surgery to the eye (Grav et al, Exp. Eye Res., vol. 31, page 119, 1979). Other biomedical applications involving hvaluronic acid and its derivatives such as hyaluronic esters as described by della Valle and Romeo, EP 0216453, 1987), concern the processes linked with tissue repair (lesions, burns).
As regards low-molecular-weight fractions, various fields of application are being successfully explored in dermatology (Scott EP0295092) B1, 1987) and pharmacology. Certain biological properties have proved to be sensitive to decreases in molecular weight and to. the function of the distribution curve characterized by various localization and dispersion indices (Mw, Mn, Mz) and by polydispersion index. For example, low-molecular-weight hyaluronic acid fractions act as potential angiogenic substances, acting on the polysaccharide's ability to increase vascularization, or intervening in the inflammatory processes as specific inhibitors of factors such as TNF (Noble et al, J. Clin. Inv., vol. 91, page 2163, 1993). Moreover, low-molecular-weight fractions of hyaluronic acid can be used in bone formation phenomena and as antiviral agents.
There are many examples of the preparation of hyaluronic acid fractions obtained by physical methods involving for example heating, ultrasounds, UV and gamma irradiation, or by enzymatic reactions using hyaluronidase (Chabreck et al., Jour. Appl. Poly. Sci., vol. 48, page 233, 1991; Rehakova et al., Int. J. Biol. Macrom., vol. 16/3 page 121, 1994); or, again, by chemical depolymerizing reactions with ascorbic acid (Cleveland et al., Bioch. Biophy. Acta, vol. 192, page 385, 1969) or by treatment with hypochlorites (Schiller et al, Biol. Chem. Hopp-Seyler, vol. 375, page 169, 1994). However, all the cited methods are flawed in some way with regard to the type of products obtained. Indeed, even though some of them do not modify the primary polymeric structure, intervening on the glycoside bonds, they are unable to generate degraded, low-molecular-weight products characterised by low polydispersion index. Indeed, it has been seen that techniques using ultrasounds or heat produce depolymerization kinetics which present asymptotic patterns. Further treatments for the same time and in the same conditions (ultrasound power and temperature) lead to the product's complete degradation.
Unlike these physical methods, depolymerization induced by the action of hyaluronidase presents certain advantages such as the efficacy of the reaction with consequent maintenance of the primary structure of the polymeric chain and control of the degradation kinetics. Observance of these parameters does not, however, guarantee high chemical yields or products characterized by low molecular weight distribution.
Lastly, the extensive action of chemical agents such as sodium hypochlorite and ascorbic acid, leads simultaneously to a loss of molecular weight and to a significant alteration in the chemical structure of the polymeric chain. Degradation derivatives with the desired molecular profile can only be obtained by a careful and controlled use of these reagents, so that the potential use of the chemical process on an industrial scale is greatly reduced.